On the formation and stability of cellulose-based emulsions in alkaline systems: Effect of the solvent quality

With amphiphilic properties, cellulose molecules are expected to adsorb at the O/W interface and be capable of stabilizing emulsions. The effect of solvent quality on the formation and stability of cellulose-based O/W emulsions was evaluated in different alkaline systems: NaOH, NaOH-urea and tetrabutylammonium hydroxide (TBAH).

Carbohydrate Polymers 286 (2022) 119257

Available online 15 February 2022

0144-8617/© 2022 The Authors Published by Elsevier Ltd This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

On the formation and stability of cellulose-based emulsions in alkaline

systems: Effect of the solvent quality

Carolina Costaa,*, Bruno Medronhoa,b, Alexandra Filipeb,1, Anabela Romanob,

Bj¨orn Lindmanc,d, Håkan Edlunda, Magnus Norgrena

aFSCN, Surface and Colloid Engineering, Mid Sweden University, SE-85170 Sundsvall, Sweden

bMED – Mediterranean Institute for Agriculture, Environment and Development, Faculdade de Ciˆencias e Tecnologia, Universidade do Algarve, Campus de Gambelas, Ed

8, 8005-139 Faro, Portugal

cPhysical Chemistry, University of Lund, P.O Box 124, S-221 00 Lund, Sweden

dCoimbra Chemistry Center (CQC), Department of Chemistry, University of Coimbra, Rua Larga, 3004-535 Coimbra, Portugal

A R T I C L E I N F O

Keywords:

Regenerated cellulose

Amphiphilicity

Dissolution

NaOH

TBAH

Urea

O/W emulsions

A B S T R A C T With amphiphilic properties, cellulose molecules are expected to adsorb at the O/W interface and be capable of stabilizing emulsions The effect of solvent quality on the formation and stability of cellulose-based O/W emulsions was evaluated in different alkaline systems: NaOH, NaOH-urea and tetrabutylammonium hydroxide (TBAH) The optimal solvency conditions for cellulose adsorption at the O/W interface were found for the alkaline solvent with an intermediate polarity (NaOH-urea), which is in line with the favorable conditions for adsorption of an amphiphilic polymer A very good solvency (in TBAH) and the interfacial activity of the cation lead to lack of stability because of low cellulose adsorption However, to achieve long-term stability and prevent oil separation in NaOH-urea systems, further reduction in cellulose's solvency was needed, which was achieved

by a change in the pH of the emulsions, inducing the regeneration of cellulose at the surface of the oil droplets (in-situ regeneration)

1 Introduction

Emulsions have a wide-spread interest and their uses range from

technical to life science applications (Walstra, 2005) During the

emul-sification process, different stabilizing and emulsifying agents have been

used Among them, surfactants, synthetic and natural macromolecules,

and particles are often used (Kronberg et al., 2014b) Synthetic

surfac-tants and polymers have played an important role, but for reasons of

sustainability and safety there is a strong trend towards alternatives

based on natural and renewable resources Here both lipids and other

macromolecules should be highlighted Carbohydrate-based systems

include novel surfactants and systems based on cellulose In this respect,

cellulose derivatives have a long tradition; both nonionic, anionic and

cationic derivatives find a broad use in emulsion formulations, such as

foods, pharmaceuticals, personal care products and water-based paints

(Murray, 2009; Sarkar, 1984; Wüstenberg, 2014) Cellulose

nano-particles, first described and made on purpose by Bengt Rånby (1951),

have rather recently been introduced as emulsion stabilizers ( Kalash-nikova et al., 2011) Their action resembles the typical Pickering effect (Pickering, 1907)

The preparation and stabilization of emulsions relies on different mechanisms depending on the emulsifier used Low molecular weight molecules, such as surfactants and lipids, have an important role in decreasing the interfacial tension between the oil and water phases thus reducing the energy needed for dispersion On the other hand, macro-molecules have a different main role; by adsorbing at the oil-water interface, they stabilize the emulsions by creating a “steric” barrier The repulsion depends on the extension of the polymer chains away from the interface and thus a favorable interaction between the polymer and the continuous medium; when droplets approach each other, a repulsion arises both due to reduced solvation and due to a decreased conformational entropy of the extending polymer chains (Kronberg

et al., 2014b) Amphiphilic polymers and nonionic surfactants, such as those with long ethylene oxide chains, can act as both emulsifiers and

* Corresponding author

E-mail address: carolina.costa@miun.se (C Costa)

1 Present address: AD-ABC, Association for the Development of the Academic Center for Research and Biomedical Training of the Algarve, University of Algarve, Campus de Gambelas, Ed 2, 8005-139 Faro, Portugal

Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

https://doi.org/10.1016/j.carbpol.2022.119257

Received 1 December 2021; Received in revised form 11 February 2022; Accepted 11 February 2022

stabilizers

Except for molecular stabilizers, it has been known for a long time

that small particles may stabilize dispersions in general, and emulsions

in particular As mentioned above, such emulsions are known as

Pick-ering emulsions after the pioneer in the field (Pickering, 1907) Cellulose

nanoparticles belong to this class of stabilizers (Salas et al., 2014) For a

good stabilization, the particles should primarily be wetted by the liquid

making up the continuous medium; the contact angle is an important

factor judging the efficiency of particle stabilization (Dickinson, 2010)

It is believed that the amphiphilic character of cellulose nanoparticles

resides in its crystalline organization at the elementary brick level, and

thus, since cellulose nanocrystals have both hydrophilic and

hydro-phobic edges, these will be preferentially wetted by water or oil,

respectively (Kalashnikova et al., 2012) In recent years there has been a

development of the so-called Janus particles (Kumar et al., 2013), i.e.,

particles with an amphiphilic character that are composed of two or

more regions with distinct physicochemical properties; these can

pro-vide efficient stabilization Whereas cellulose derivatives are well

established as emulsion stabilizers and various nanocelluloses have been

receiving an increasing interest, we believe that molecularly dissolved

cellulose should be also considered as an efficient natural candidate for

emulsion formation and stabilization (Costa, Medronho, et al., 2019)

Cellulose dissolution has been an active field for a long time but in

recent years there has been a strongly renewed interest, not only driven

by UN's agenda for a sustainable development, but also as a result of new

emerging applications of cellulose Many solvents have been developed

throughout the years and are well described in literature, ranging from

ionic liquids and organic systems to water-based ones (Medronho &

Lindman, 2014) With the goal of using pristine cellulose for emulsion

preparation and stabilization, the most direct way would consist in

having unmodified cellulose soluble in one of the constituent liquids

since, otherwise, solvent removal would require additional laborious

steps in the manufacture Cellulose is not soluble in water or oil but can

be dissolved in certain aqueous systems at high or low pH's; this can be

ascribed to an ionization effect by protonation or deprotonation giving

cellulose a polyelectrolyte-like character (Bialik et al., 2016; Lindman

et al., 2010; Lindman et al., 2017) In addition, it has been observed that

the dissolution can be facilitated by amphiphilic additives or by using

acids or bases with organic counterions (Alves et al., 2015; Alves et al.,

2016b; Medronho & Lindman, 2014, 2015) The reason for this is

ex-pected to have a marked contribution from cellulose's amphiphilic

character and concomitant role of hydrophobic interactions Therefore,

reducing the hydrophobic interactions by having acids or bases with

“hydrophobic” ions or adding substances known to reduce hydrophobic

interactions, are important aspects to consider when developing novel

solvent systems and rationalizing cellulose's dissolution mechanism

Urea is a common example of such an additive Its weakening effect on

hydrophobic interactions does not only significantly reduce the

ten-dency of surfactant self-assembly or affect protein and DNA stability, but

also enhances cellulose dissolution (Lindman et al., 2021)

Although cellulose dissolution in aqueous media is a central issue for

emulsions, some aspects related to its dissolution and self-assembly

mechanisms are still debatable (Lindman et al., 2021) Nevertheless,

its clear amphiphilic features are expected to drive cellulose to self-

assemble at oil-water interfaces and be able to stabilize emulsions As

mentioned, recent studies have found that cellulose nanocrystals display

amphiphilic properties and can give stable emulsions It was further

observed that nanocrystals with low surface charge favor the stability of

emulsions, suggesting that the amphiphilic nature of cellulose is the

main driving force for the stabilization (Kalashnikova et al., 2012)

Molecularly dissolved cellulose is likely to behave closer to typical

cellulose derivatives, namely the cellulose ethers, widely used

commercially as emulsion stabilizers (Hon, 2001; Jedvert & Heinze,

2017; Murray, 2009; Seddiqi et al., 2021) However, native cellulose has

been much less explored due to the mentioned dissolution limitations In

our previous works, we have been focusing on acidic water-based

solvents for cellulose molecular dissolution and emulsion formation (Costa et al., 2021; Costa, Medronho, et al., 2019; Costa, Mira, et al.,

2019; Medronho et al., 2018) We have shown that molecular cellulose dissolved in aqueous solutions of phosphoric acid (H3PO4 (aq.)) adsorbs

at the oil-water interface (Costa, Mira, et al., 2019) For instance, the interfacial tension between oil and H3PO4 (aq.) solution used to dissolve cellulose was found to be lowered by the presence of cellulose and this decrease was similar in magnitude to that displayed by nonionic cellu-lose derivatives in water (Wüstenberg, 2014) From these results, it can also be rationalized that the amphiphilicity of cellulose derivatives is, to

a large extent, due to the hydrophobic cellulose backbone (Lindman

et al., 2021) Furthermore, the aging of the cellulose-stabilized emul-sions formed in that same solvent was evaluated; emulemul-sions were found

to be short-lived, as oil was separating from the emulsions and floating

to the top within 24 h However, by adding water to the dispersions as a second step during emulsification, the properties of the emulsions changed dramatically, and there was no evidence of oil separation over one year of storage (Costa, Mira, et al., 2019) This effect was attributed

to a decrease in cellulose solvency with water, thus acting as an “anti- solvent” The decreased solvency promotes a greater affinity for the oil- water interface, which, in turn, may lead to the remarkable emulsion stability against macroscopic phase separation

With the present work we wanted to extend the knowledge on the emulsification capabilities of dissolved cellulose to another type of aqueous non-derivatizing solvents, the alkaline, and moreover, under-stand how the solvent quality (i.e., cellulose dissolution state) may affect the migration of cellulose molecules to the O/W interface, and thus, emulsion stabilization The selected alkaline (aqueous-based) solvents were NaOH (Alves et al., 2015; Alves et al., 2016a), NaOH-urea (Alves

et al., 2016a; Cai & Zhang, 2005) and TBAH (Alves et al., 2015; Alves

et al., 2016a) Recent research suggests that in the two latter cases, cellulose dissolves down to the molecular level whereas in neat NaOH (aq.) cellulose aggregates persist (Alves et al., 2015; Alves et al., 2016a) Different methods were used to infer on the effect of the solvent system properties on emulsion formation and stability These included tensi-ometry, turbidimetry, optical microscopy, laser diffraction, micro-rheology and carbohydrate quantification

2 Materials and methods

2.1 Materials

The cellulose source was microcrystalline cellulose (Avicel PH101, Sigma Aldrich) with a weight-average molecular weight (Mw) of

≈62,000 g/mol and a polydispersity index of 3.85, as determined by size exclusion chromatography NaOH (99.2% purity), dodecane (100%), hydrochloric acid (HCl-37%) and sulphuric acid (H2SO4–95%) were purchased from VWR chemicals Urea (≥99% purity), TBAH (40 wt% aqueous solution), α-D-glucose (anhydrous, 96%), orcinol (97%) and calcofluor white stain, were obtained from Sigma Aldrich MilliQ water was used in the preparation of all aqueous solutions Sulfate-latex par-ticles (8 w/v% aqueous solution) were purchased from ThermoFisher Scientific

2.2 Cellulose dissolution

Solutions of 0.5 wt% cellulose were prepared with three different alkaline aqueous solvents: 7 wt% NaOH (1.9 M), 7 wt% NaOH-4 wt% urea (1.9–0.75 M) and 40 wt% TBAH (1.5 M) The solutions were pre-pared following an adapted procedure (Pereira et al., 2018) Briefly, the cellulose powder was added to the alkaline solvents at room tempera-ture (20–21.5 ◦C) and all samples were vigorously stirred for 1 min with

a vortex The sample with TBAH was kept under mild stirring at room temperature overnight, until the cellulose was completely dissolved The samples with NaOH and NaOH-urea were placed in the freezer at − 30 ◦C for 10 min, removed and then vigorously stirred (while cold) for another

Carbohydrate Polymers 286 (2022) 119257

minute This way, the suspensions become more homogeneously mixed

and stable until the complete freezing of the samples The solutions were

kept in the freezer overnight and then thawed at room temperature

Clear solutions were obtained after this procedure and dissolution was

confirmed by polarized light microscopy

2.3 Cellulose emulsions and suspensions

O/W emulsions were prepared by dispersing 10 wt% dodecane in the

different cellulose solutions for 5 min using an Ultra-Turrax mixer at the

speed of 16,000 rpm For a total mass of 25 g emulsion, small beakers of

40 ml and a diameter of 4 cm were used to perform the homogenization

of all different alkaline systems Subsequently, emulsions were prepared

for each solvent system by decreasing the pH of the continuous phase by

the addition of precise amounts of HCl solution (1–3 ml, i.e.,

0.012–0.036 mol of acid per addition) This procedure allows to follow

the changes occurring in emulsion formation and stability upon the

progressive decrease of cellulose solubility, for each solvent system The

highest amount of acid added (0.036 mol) was observed to decrease the

pH of the NaOH-based emulsions below 13.5, which is the pKa value

reported for cyclodextrins (a good cellulose model candidate) (

Gaida-mauskas et al., 2009) Below this pH, cellulose is expected to have a

lower charge density For TBAH, even though used with a lower

molarity than NaOH, the pH was still higher than 13.5, which might be a

result of its superior basicity For these emulsions post-regenerated with

acid, a second step in the emulsification was considered; for each ml of

HCl solution added, 3 extra min of stirring with the Ultra-Turrax were

performed The HCl solution was added drop-wise while emulsions were

stirring at 16000 rpm, to ensure a fast spreading of the acid to the whole

system and to avoid, as much as possible, heterogeneous cellulose

regeneration Following this procedure, no major clumps of regenerated

cellulose were visually observed Control samples of the different

re-generated systems were made following the same procedure as

previ-ously described, but without the addition of the oil This resulted in

simple suspensions of regenerated cellulose in the different alkaline

solvents

2.4 Turbidimetry of cellulose suspensions

The turbidity of the regenerated suspensions of cellulose in the

different alkaline solvents was determined using a Hach RATIO/XR

43900 turbidimeter, equipped with a tungsten lamp The turbidity

readings were performed directly on the instrument, using a

nephelo-metric turbidity unit (NTU) scale, based on white light (400–680 nm)

and 90◦ incident angle The measurements were performed with

matched Hach glass sample cuvettes with screw caps at room

temper-ature (20–21.5 ◦C) and performed immediately after preparation

2.5 Diffusion wave spectroscopy of cellulose emulsions

A RheoLab instrument (LS Instruments AG, Fribourg, Switzerland),

equipped with the echo technology, was used for diffusing wave

spec-troscopy (DWS) analysis in the transmission mode DWS is an advanced

light-scattering technique that accesses the microrheology of highly

turbid samples by approximating the propagation of light to the

diffu-sion equation; a detailed theoretical framework of this method can be

found elsewhere (Niederquell et al., 2012; Reufer et al., 2014) Briefly,

an emulsion contains oil droplets that perform Brownian motions, which

are sensitive to the rheology of the system When a laser source

illu-minates the sample loaded in a cuvette, the photons will be scattered

multiple times by the oil droplets Intensity fluctuations are detected

when the scattered photons emerge at the opposite side of the incident

light These intensity fluctuations are then used as a probe to

charac-terize the rheological properties of the sample A glass cuvette made of 5

mm in thickness (L), was used and all the samples were vigorously mixed

for 120 s, using a vortex mixer, and immediately measured at 25 ◦C The

measurement time was set to 120 s with additional 60 s of echo duration Three individual runs for each sample were performed, and the

esti-mated error was below 5% Before measurements, the l* calibration (being l* the transport mean free path in the medium) was performed

using as reference standard an aqueous solution composed of 0.55 wt%

of sulfate-latex particles of mean diameter of 500 nm, which guaranteed

a deviation of less than 20% between the CR of the sample and the

standard The calibration was performed using the Rheolab l*

calibra-tion mode at 25 ◦C and a glass cuvette of the same thickness as used for the samples

2.6 Optical microscopy of cellulose suspensions and emulsions

Samples were observed on a Leica DMRX light microscope Typi-cally, a small droplet of the dopes was placed on a glass slide and covered with a cover slip Samples were analyzed by transmitted light polarization using crossed polarizers, at magnifications of 10× and 20× Pictures were captured using a Leica DFC 320 camera with 5 megapixels Samples were also stained with calcofluor white and observed in an Axioimager Z2 (Carl Zeiss) fluorescent microscope and recorded with the b/w camera Axiocam HRm (Carl Zeiss) Images were acquired and processed using the Axiovision 4.8 software (Carl Zeiss) and ImageJ (open source software from the National Institutes of Health, USA)

2.7 Droplet size measurements of cellulose emulsions

The median droplet diameter (D50) of the cellulose-based emulsions was determined by laser diffraction using a Mastersizer Hydro 2000SM instrument, using a He-Ne gas laser red light with a beam wavelength of

633 nm and a LED blue light with a wavelength of 466 nm (Malvern Instruments, UK) For these measurements, a small volume of the emulsions was added drop-wise into a sample dispersion unit (at 2000 rpm), filled with water, until an optimum measuring value was reached

by the program (i.e., an indirect indication of the number of drops)

2.8 Interfacial tension of cellulose solutions/dodecane

The force tensiometer K20 (Krüss) equipped with a Wilhelmy plate, was used to measure the interfacial tension (IFT) between the different cellulose-alkali solutions and dodecane Cleaning of the plate and bea-kers was done by flushing the materials with acetone, ethanol and water,

in that exact order, and then the materials were dried and placed in a plasma cleaner for 3 min This procedure was repeated before each measurement, followed by the plate calibration, which was performed

by measuring the surface tension between air and water (values between

ca 72.5–73 mN/m were typically obtained) Measurements were taken every 5 min during a period of 60 min for each sample

2.9 Carbohydrate content in the emulsion serums

The carbohydrate content of the serums of the post-regenerated NaOH-based emulsions was determined using the Orcinol method, following the SCA-F W 15:77 protocol (SCA R&D Centre, 2011) The Orcinol method is based on a color reaction of carbohydrates with 0.2% orcinol reagent dissolved in concentrated sulphuric acid A calibration curve was obtained by preparing calibration solutions of D-glucose with 0.1 g accuracy, and the concentration of carbohydrates in the sample of interest was then calculated from the equation of this calibration curve The absorption of the samples was measured using a UV-1800 Shimadzu UV–vis spectrophotometer at a wavelength of 480 nm

C Costa et al

3 Results and discussion

3.1 Effect of solvent quality on the adsorption of cellulose at the oil-water

interface

An important goal of this work was to better understand the

mech-anisms behind the adsorption and interfacial activity of cellulose

mol-ecules at the oil-water interface, and to determine how the solvent

quality affects such behavior This was done by using alkaline water-

based solvents with different features, such as polarity and

amphiphi-licity, in order to perceive what are the most suitable dissolution

con-ditions for cellulose, which successfully contribute to stabilize O/W

emulsions The solubility of cellulose is increased in the following order:

NaOH < NaOH-urea < TBAH (Alves et al., 2016a) The interfacial

ac-tivity of cellulose was assessed by measuring the IFT between the oil-

phase (dodecane) and the different solvents containing cellulose

(Fig 1A) In the NaOH-based solvents, the interfacial tension decreases

with the presence of cellulose, with the decrease being more pronounced

in the solvent with an intermediate polarity (NaOH-urea) On the other

hand, with the TBAH solvent it is more intriguing to characterize the

interfacial behavior of cellulose, since the solvent itself gives a quite low

IFT due to the presence of the amphiphilic cation TBA+; in fact, cellulose

seems to affect the IFT slightly in the opposite way, which may be

ascribed to a competition between the TBA+ions and cellulose for the

interface

In discussing the interfacial behavior of cellulose, the complications

due to adsorption of solvent constituents at the interface must be

accounted for An amphiphilic ion like TBA+is expected to have a sig-nificant adsorption at oil-water interfaces as it is clearly confirmed by our observations (Tamaki, 1967) This makes inferences on cellulose adsorption from such solutions difficult; the slight increase in IFT sug-gests, however, competition with TBA+for the interface and some cel-lulose adsorption Urea has been found to give a weak decrease of the IFT in the related decane-water system (Jones, 1973) In our present work, no major differences were noted between the NaOH and NaOH- urea system (Fig 1A, grey curve “NaOH-based solvents”) Therefore, the downward shift of the IFT curve from the NaOH to the NaOH-urea system may be ascribed to the enhanced dissolution of cellulose in the latter case, where more individual cellulose molecules may be available

to adsorb at the interface more efficiently To conclude about the interfacial behavior of cellulose from the lowering of the IFT, it is clear from the results for the two NaOH solvents that cellulose shows a sig-nificant adsorption at the oil-water interface; this is in line with the results for acidic solvents and also in line with an amphiphilic character

of cellulose For the TBAH case, the amphiphilic properties of the solvent cation preclude clear conclusions from IFT measurements; direct mea-surements like ellipsometry or neutron reflection could be helpful In lack of clear information, it can be noted that in an environment of amphiphilic ions like TBA+we do not expect a major driving force for cellulose adsorption

3.2 Emulsification ability of cellulose as a polymeric emulsifier

The study of the IFT gives a basis for investigations of the ability of

Fig 1 A) Interfacial tension between dodecane and the alkaline solvents (grey curves) and respective cellulose solutions (black curves); the grey dotted curve

“NaOH-based solvents” represents both alkali solvents: NaOH and NaOH-urea B) Light microscopy micrographs of the resultant emulsions stabilized by cellulose in the NaOH-based solvents C) Visual appearance of the vials; stability against oil separation in the different alkali-based emulsions

Carbohydrate Polymers 286 (2022) 119257

cellulose to adsorb at oil droplets in O/W emulsions and thus to stabilize

the emulsions O/W emulsions were prepared with the different alkaline

solvents, and their behavior was first followed (visually) with time As it

is observable from the photos in Fig 1C for the TBAH system, already 5

min after homogenization the oil was almost completely separated from

the emulsion, with only a few droplets being left in the emulsion; these

droplets were separating in 24 to 48 h In the case of NaOH-based

sol-vents, visual oil separation was only noticed after one week This

phe-nomenon was, however, more pronounced in the solvent system

composed of NaOH-urea (Fig 1C)

Stabilization of dispersions by polymers can be discussed mainly

from two effects; the degree of polymer adsorption and the conformation

of the polymer chains at the surface (Kronberg et al., 2014a) A

dis-cussion can be fruitfully based on solvency effects A poor solvency leads

to poor dispersion stability since the polymer layer will have limited

thickness On the other hand, a very good solvency leads to lack of

stability because of low adsorption Therefore, optimal stabilization is

attained at intermediate solvency conditions (if the polymer has an

amphiphilic character this is favorable for adsorption and stability)

These arguments are based on the adsorption of individual polymer

molecules from solution If the polymer is not dissolved to the molecular

level but is aggregated, a more complex behavior can be expected

including the presence of polymer aggregates or “lumps” on the surface;

then additional stabilization can be achieved due to the presence of

relatively thick polymer layers

The emulsions morphology (Fig 1B) and droplets median sizes (D50)

were obtained by light microscopy and laser diffraction, respectively

The droplet sizes were only measured for the NaOH-based systems

Because both NaOH-based emulsions had a bimodal distribution,

vol-ume and number of distributions were both considered for the

calcula-tion of D50 Approximate values were obtained for both emulsion

systems, 18–20 μm in volume and 3–5 μm in number, with spans of 1.4

to 3 (see also the size distributions in Fig S1) Looking at the

micro-graphs in Fig 1B, small micrometer droplets in the approximate size as

given by the number distribution, can be actually observed which seems

to be in good agreement

3.3 Effect of solvent quality on cellulose regeneration at the oil-water

interface

In acidic aqueous solvents, emulsions stabilized by cellulose

mole-cules are short-lived, but adding water in a second step of the

emulsi-fication process induces cellulose regeneration at the interface, and

provides outstanding long-term stability (one year or even more) (Costa,

Medronho, et al., 2019; Costa, Mira, et al., 2019) In the present study,

an alternative approach was followed: cellulose solubility was

progres-sively decreased by decreasing the solution pH as second step of the

emulsification procedure The changes occurring in the emulsions upon

pH decrease were followed by visual inspection In Fig 2, the first vial

for each solvent system represents the neat emulsions without any acid addition, i.e., the emulsions stabilized by cellulose adsorbed at the O/W interface Upon the progressive in-situ regeneration, data strongly sug-gests that the adsorption of cellulose at the interface is affected by the solvent quality In the NaOH-based systems, the picture is similar, but there are a few differences worth noting For both cases, the stability against oil separation increases, and opposite to what happened before regeneration for the NaOH-urea system, now the stability against oil separation matches the one for the NaOH system This can be explained

by a change in the physical state of the cellulose molecules and the formation of a soft-hard cellulose “shell”, a more robust mechanical barrier to prevent oil escaping It is also noticed that the stability against creaming improves upon further pH decrease This means that more cellulose is being regenerated and taking part in stabilizing the oil droplets, either by adsorbing to the interface or contributing to the re-generated cellulose network in the continuous phase Both emulsion systems present clear serums (depleted of oil droplets) at the bottom of the vials, which indicates that cellulose steric stabilization is not strong enough at the concentration used for this study (i.e., 0.5 wt%) However, the serum of the NaOH-urea system has a smaller volume compared to the NaOH system, indicating an improvement in creaming stability In the NaOH-urea system, on the second vial (first acid addition) it is noteworthy that part of the regenerated cellulose seems to be settling after a while (i.e., 24–48 h), but it cleared out after shaking and aging again This cellulose sedimentation is probably due to a weak network of cellulose that starts regenerating in the bulk, and existing mostly in the form of flocks due to the low concentration used This is sometimes also seen in the NaOH-based system, but less evident than for NaOH-urea solvent

The case of TBAH is strikingly different from the NaOH-based sol-vents The first acid addition does not seem to have an effect on cellulose adsorption to the interface; for the second acid addition there seems to

be a small layer of oil on top, with an emulsion in the middle and a clear serum at the bottom Further analysis by visual inspection with time and the fluorescence microscopy reveals that oil droplets are actually

“trapped” in a viscous network of regenerated cellulose, which eventu-ally will separate and part of the regenerated cellulose will sediment covering the serum part This means that, when dissolved in TBAH, cellulose regenerates in the bulk rather than at the interface of the emulsions To get a better insight into the differences between regen-erating cellulose in the different alkali systems and also to compare the regeneration of cellulose in the bulk versus in the presence of oil drop-lets, cellulose suspensions were analyzed by turbidimetry, and later, both suspensions and emulsions were analyzed by fluorescence microscopy

In Fig 3, it can be observed that the suspensions of both NaOH-based solvents are not visually very different, but the turbidity measurements tell otherwise For the highest amount of acid added, a substantial decrease in turbidity for the suspensions in NaOH-urea is observed,

Fig 2 Photographs of short-time stability tests (7 days) of post-regenerated emulsions For each solvent system, equal moles of acid were added progressively from

the left to the right vials The first vial in each solvent system represents the emulsion without acid (only dissolved cellulose)

C Costa et al

which can be translated into a different organization of the cellulose

molecules upon regeneration from a solvent with an intermediate

po-larity, and possibly more amorphous structure, as it has been shown by

Alves et al (2016a) It is also possible to see that after 48 h of aging of

the suspensions, the stability against sedimentation increases in the

order: NaOH < NaOH-urea < TBAH (Fig 3) This observation could

possibly relate to the slightly better stability against creaming of the

post-regenerated emulsions in the NaOH-urea solvent (3rd vial on Fig 2)

given that, from droplet size measurements, no significant differences

are detected for both NaOH-based systems (i.e., D50 was calculated to be

16–17 μm from the volume distributions, and 6 μm from the number

distributions, with spans of 1.2 to 1.6; see also the size distributions in

Fig S1) It was also observed, that below a certain pH (slightly below the

pKa value), the sizes of the emulsions are no longer affected by pH and

cellulose is expected to be more or less irreversibly adsorbed at the

emulsion interface For the TBAH system, it is noteworthy that

precip-itation of the cellulose molecules is only observed upon the addition of

three times more acid than for the NaOH-based systems This can be

reasoned by the higher basicity of the TBAH, which possibly leads to a

higher charge density of the cellulose chains Consequently, a higher

amount of acid is needed to neutralize enough charges and trigger

cel-lulose regeneration (Bialik et al., 2016)

When comparing the regeneration of cellulose in TBAH systems in

the presence or absence of oil, no apparent difference is seen (Fig 4, 3rd

column) Fluorescence microscopy revealed a similar structure of the

regenerated cellulose network with no oil droplets stabilized by

cellu-lose, since no fluorescence is detected around the droplets seen in the

picture (the fluorescence arises from the bulk) On the other hand, for

both NaOH systems (Fig 4, 1st and 2nd columns), the oil droplets reveal a

thin fluorescent layer of cellulose surrounding them and a network of

cellulose in the continuous phase Therefore, we can conclude that

cel-lulose molecules stabilize emulsions in a very similar way as its celcel-lulose

derivatives do: a) by reducing the interfacial tension, arising from the

amphiphilic character of the cellulose backbone; b) adsorption of a

cellulose layer that provides steric repulsion; and c) formation of a

network on the continuous phase

3.4 Carbohydrate content in the serums of the emulsions

In our previous work, it was found that the stabilized acidic

emul-sions exhibited creaming after 24 h of storage, leaving a clear liquid

fraction “serum” (depleted of droplets) at the bottom This was attrib-uted to the aggregation of droplets due to the neutralization of the cel-lulose charges when dissolved at extreme pH conditions (Costa, Medronho, et al., 2019; Costa, Mira, et al., 2019) Similar aggregation and creaming phenomena of the emulsions are expected to occur in the alkaline-based aqueous solvents If cellulose is diffusing from bulk so-lution to the emulsion interface upon pH decrease, then the carbohy-drate content in continuous phase (serum) is expected to decrease In Table 1 it is possible to observe that the progressive addition of HCl makes the continuum media depleted from cellulose On the other hand,

as the degree of dissolution is enhanced by the presence of urea, it is not surprising that a higher cellulose concentration is still found in the serum of the NaOH-urea system in comparison to the neat NaOH sol-vent Since cellulose is expected to be more aggregated in the NaOH system than in NaOH-urea, larger particles are expected to adsorb at the emulsions interface in the former solvent while molecularly dissolved cellulose (or close to it) will adsorb in the latter case, thus leaving more cellulose available in the bulk When the pH decreases substantially, the solvent quality becomes poorer and the majority of cellulose is expected

to be found at the emulsions interface, regardless the NaOH system These results clearly demonstrate that the dissolution degree of cellulose together with the solvent quality (here affected by the pH) are funda-mental parameters to understand the emulsion formation and stability

in these strongly alkaline systems Note that no determination of car-bohydrate content was performed in the TBAH system due to the poor stability of the emulsions formed

3.5 Microrheology and stability of emulsions

The rheological characterization of the emulsions is of major importance since it provides information on their physical stability and

on the molecular features of the continuous phase Therefore, the microrheological properties of the regenerated NaOH-based emulsions systems were extracted by DWS transmission measurements; the com-plex viscosity (η*), storage (G′) and loss (G′′) moduli are displayed in Fig 5a and b

The first observations are that the emulsion systems have similar η* This agrees with the rather similar average size observed in both NaOH- systems and similar content of sugars in the continuous phase (see Table 1) Since the crossover occurs at high frequencies, we can assume that the samples are essentially viscous From this crossover, the

Fig 3 Turbidity of the cellulose suspensions in the

different alkaline solvents in freshly prepared sam-ples; images of the vials correspond to the respective solvent in the curves, i.e., NaOH, NaOH-urea and TBAH, from the top to the bottom The numbers on top of the vials correspond to the HCl (37 wt%) vol-ume additions The samples with 3 ml acid addition were aged for 48 h and the results are shown in the green square (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Carbohydrate Polymers 286 (2022) 119257

relaxation time, τ, was estimated as ca 8.83 × 10− 5 s and 4.62 × 10− 5 s

for NaOH and NaOH-urea systems, respectively As discussed, urea is

known to improve cellulose dissolution; the decrease in this short-time

relaxation with the addition of urea in the solvent system can suggest

that the degree of cellulose dissolution may affect the dynamics and

relaxation mechanisms that occur at the microscale (high frequencies),

such as dissipation mechanisms

DWS analysis performed in the transmission mode also enables to

infer about the emulsions stability by evaluating the intensity

correla-tion funccorrela-tion (ICF) decay over the aging time of the emulsion The ICF

decay consists on the normalized temporal fluctuations of the scattered

light intensity and is sensitive to the concentration and size of the tracers

(i.e., oil droplets) For larger and/or less concentrated oil droplets, the

ICF decay will occur at longer lag times On the other hand, a decay to

short lag times is expected for more concentrated droplets and/or

smaller and comparatively rapid ones Consequently, a shift of the ICF

decay to long lag times, over the aging time of the emulsions, can reflect

the onset of destabilization mechanisms, such as Ostwald ripening,

creaming, flocculation or coalescence (Medronho et al., 2018)

There-fore, the stability of the regenerated NaOH-based emulsions with time

was followed by DWS in the transmission mode during 48 h and the differences in the normalized ICF vs lag time during sample aging were evaluated (Fig 6a and b)

A shift towards higher lag times in the ICF decay was obtained for both samples tested The moment when the ICF equals 1 corresponds to the moment at which no scattered light is detected, indicating the roughly complete physical separation between the oil and water phases, and it occurred after 30 min for the NaOH sample and 60 min for the NaOH-urea To better compare the onset of destabilization, the t2/3 parameter, corresponding to the lag time at which the ICF curve decays

to 2/3 of its initial value, was determined for both samples and plotted

as a function of the aging time (Fig 7a)

The onset of destabilization occurs at approximately the same time for both samples, since the t2/3 parameter starts to increase after ca 27 min for the NaOH emulsion and ca 30 min for the NaOH-urea emulsion The enhanced dissolution of cellulose in the NaOH-urea system allows for a dissolution state closer to the molecular level, presumably making cellulose molecules capable of diffusing and adsorbing more efficiently

at the oil-water interface, thus enhancing its stability and delaying the complete phase separation

The transport mean free path, l*, corresponds to the average distance

a photon needs to travel before completely losing its direction (randomization) It is sensitive to the concentration of the scattering particles present in the solution and, for this reason, it can also contribute to elucidate the mechanisms involved in the emulsion

destabilization processes The l* parameter of a given sample (l* sample) is

also related to its count rate (CR sample), through the equation (Medronho

et al., 2018):

l*sample=l*ref×CR sample

CR ref

where the CR is defined as the average measure of the photons arriving

in the detector; CR ref and l*ref are estimated by measuring a calibration

standard and the CR sample is obtained during the ICF measurement of the

Fig 4 Fluorescence micrographs of the cellulose suspensions (on top) and emulsions (on the bottom) of each alkali system All samples were stained with calcofluor

white (3 ml HCl solution addition for both emulsions and suspensions)

Table 1

Carbohydrate content estimated in the continuous medium (serum) after acid

addition by the Orcinol method (SCA R&D Centre, 2011) Images show the

colorations acquired by the serums upon reaction with the orcinol reagent;

lighter colors mean a reduction in carbohydrate content

C Costa et al

sample As can be observed in Fig 7b, a gradual decrease over time of

both parameters is obtained for the NaOH-urea system Similar profile

was observed for the neat NaOH solvent (data not shown) These results

suggest that the decrease in the l* parameter might be due to a decrease

in the number of scattering events over time This further indicates that

the emulsion's destabilization might result from creaming, since the

migration of the oil particles to the upper layer of the solution during the aging would result in a decrease of the scattering events, leading to a

decrease in l* and CR In our previous work with stabilized acidic

emulsions (Costa et al., 2021; Medronho et al., 2018), the l* and the t2/3

parameters exhibited a similar trend but increasing with the aging time This similar trend might reflect the occurrence of the Ostwald ripening

Fig 5 Viscoelastic parameters determined by DWS at 25 ◦C of the regenerated NaOH (grey circles) and NaOH-urea (black circles) emulsion systems: (a) complex viscosity (left); (b) G′(full symbols) and G′′(empty symbols)

0 0.2 0.4 0.6 0.8 1

10 -5 10 -4 10 -3 10 -2 10 -1 10 0 10 1

0 min

12 min

24 min

30 min

g 2

Lag time (s)

0 0.2 0.4 0.6 0.8 1

10 -5 10 -4 10 -3 10 -2 10 -1 10 0 10 1

0 min

6 min

18 min

30 min

50 min

g 2

Lag time (s)

Fig 6 Normalized ICF curves obtained for the NaOH (a) and NaOH-urea emulsions (b) at different times

10 -1

10 0

10 1

t 2/

Incubation time (min)

0 200 400 600 800 1000

0 200 400 600 800 1000

Incubation time (min)

Fig 7 (a) t2/3 Parameter for the NaOH (grey line) and NaOH-urea (black line) emulsions over time (b) Evolution of the count rate (black line) and l* (grey line) parameters for the NaOH-urea sample

Carbohydrate Polymers 286 (2022) 119257

phenomenon since, at a constant volume fraction of oil, there is a

concomitant increase of the particles size with a decrease in their

number density, leading to larger values of l* Nevertheless, we expect to

further elucidate on the destabilization mechanisms of cellulose

stabi-lized emulsions in forthcoming works

4 Conclusions

The formation and stability of cellulose-based O/W emulsions in

different alkaline systems were evaluated focusing on the effect of

sol-vent quality Cellulose was found to decrease the IFT substantially and

this effect is ascribed to the cellulose migration to the oil-water

inter-face In this respect, the TBAH (aq.) was found to induce the lowest IFT

itself without a significant contribution from cellulose It is suggested

that this effect is due to the amphiphilic behavior of the TBA+cation and

its capacity to compete with cellulose for the oil-water interface The

practical consequence of this result is that when oil is added and

emulsions formed, their stability is very poor (due to the lack of cellulose

at the interface) as demonstrated by fluorescence microscopy and aging

tests On the other hand, the addition of urea to NaOH (aq.) is expected

to improve cellulose dissolution and therefore more cellulose in the form

of molecules will be available to adsorb at the emulsion interface (as

confirmed by the carbohydrate quantification), stabilizing it more

effi-ciently than the neat NaOH system These results found support from the

microrheology assays where, in addition, the destabilization mechanism

of the emulsions formed in the NaOH systems is suggested to occur via

creaming In conclusion, the adsorption behavior of cellulose is

depen-dent on the solvent quality Intermediate solvency conditions are

favorable for cellulose adsorption at the interface, which is in line with

the typical behavior of amphiphilic polymers A very good solvency of

cellulose (in TBAH) leads to a lack of stability due to low adsorption of

the molecules, and oil will quickly separate from the emulsion

Inter-mediate solvencies allow cellulose to stabilize emulsions in a short-term

To achieve long-term stability, further solvency decrease is needed after

oil dispersion in the cellulose solution, what we call the in-situ

regen-eration The rate of droplets coalescence is then dramatically reduced

and emulsions show a remarkable stability against oil-separation

Supplementary data to this article can be found online at https://doi

org/10.1016/j.carbpol.2022.119257

CRediT authorship contribution statement

Carolina Costa: Conceptualization, Investigation, Writing – original

draft, Writing – review & editing Bruno Medronho: Conceptualization,

Writing – review & editing, Supervision Alexandra Filipe:

Investiga-tion, Writing – review & editing Anabela Romano: Writing – review &

editing, Supervision Bj¨orn Lindman: Conceptualization, Writing –

re-view & editing Håkan Edlund: Writing – rere-view & editing,

Supervi-sion Magnus Norgren: Conceptualization, Writing – review & editing,

Supervision

Declaration of competing interest

The authors declare that they have no known competing financial

interests or personal relationships that could have appeared to influence

the work reported in this paper

Acknowledgments

Is with great honor that we dedicate this paper to the memory of our

dear friend Prof Maria Graça Miguel This research was funded by the

Swedish Research Council (Vetenskapsrådet), grant number 2015-

04290 Bruno Medronho acknowledges the financial support from the

Portuguese Foundation for Science and Technology (FCT), via the

pro-jects PTDC/ASP-SIL/30619/2017, UIDB/05183/2020 and the

researcher grant CEECIND/01014/2018 The Coimbra Chemistry Centre

(CQC) acknowledges FCT through the Project UID/QUI/00313/2020 The Light Microscopy Unit of CBMR-UAlg is also acknowledged; the Microscopy Unit is partially supported by national Portuguese funding FCT via PPBI-POCI-01-0145-FEDER-022122

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